Project Summary The RIght Open reading frame Kinase 3, or RIOK3, is an understudied member of the atypical protein kinase family whose function has not been firmly established. On the NIH Illuminating the Druggable Genome/ Pharos resource, RIOK3 currently boasts only eight publications, eight possible protein interaction partners, and one small molecule ligand. We recently found, quite unexpectedly, that RIOK3 plays an important role in the cellular defense against viral infection. The goal of the research proposed here is to understand how RIOK3 mediates cellular immune defenses and to gain insight into how this kinase could be targeted for antiviral and/or anti-inflammatory therapies. Results will be made available on the IDG/Pharos web resources. Rift Valley fever virus (RVFV) is an emerging human and animal pathogen that can cause serious disease and death in humans and domestic livestock. There is currently no proven, licensed treatment or vaccine for RVFV infections. We found that upon infection with RVFV, mammalian cells dramatically upregulate transcription of RIOK3. Furthermore, shortly after infection, a new pattern of RIOK3 mRNA processing occurs, resulting in abundant alternatively spliced RIOK3 mRNAs that code for a truncated peptide lacking the putative RIOK3 kinase domain. Preliminary experiments showed that siRNA knockdown of RIOK3 enhanced viral replication, and overexpression of RIOK3 diminished viral replication, demonstrating that RIOK3 plays an antagonistic role to RVFV replication. We also observed that production of interferon-? after infection with virus is diminished in cells in which RIOK3 has been knocked out, but the mechanism for these effects is unknown. We hypothesize that RIOK3's kinase activity is key to mounting a cellular immune response to infection and that it is an essential component to a signaling cascade that culminates in an antiviral state. The goal of the proposed research is to investigate the function and molecular interactions of the full-length and virally-induced truncated isoform of RIOK3. Using RIOK3 knockdown and CRISPR knockout cells, we will express the full length, truncated, and kinase-null versions of RIOK3 in transfected cells. We will measure virus propagation as a function of the different isoforms, and we will measure the cellular antiviral response via known response markers. We will also make use of potential inhibitors of RIOK3 identified in Pharos from kinome screens to assess effects on inflammatory/antiviral markers. In parallel, we will investigate the interacting partners of RIOK3 using co-immunoprecipitation experiments and phosphoproteomics. The results of this pilot work will form the basis for a larger NIH application that will describe a potential new druggable kinase target with applications to antiviral therapy and/or the modulation of the cellular inflammatory response.